Suzanne de Bruijn

190 Chapter 4 SUPPLEMENTARY MATERIALS AND METHODS SNP genotyping The RP17-locus was previously established using polymorphic markers selected from the Généthon genetic map, that were genotyped in 23 individuals from index family NL1. 1 Subsequently, we collected DNA from 27 individuals (18 affected and 9 unaffected subjects) from the fourth generation of the family. SNP-genotyping was performed on these 27 DNA samples from generation four, and for 36 individuals (17 affected and 10 unaffected subjects and 9 spouses) from the second and third generation using the HumanCore-24V.1.0 array (Illumina). The RP17-locus was further refined by determining phase in a two-parent-sib dataset. Exome and Genome sequencing Index family NL1; Whole exome sequencing (WES) was performed for three affected individuals from different branches of the family. Exome enrichment was performed using the Aligent SureSelect Human All Exome V5 kit following manufacturer’s instructions. Subsequently, WES was executed on an Illumina HiSeq2000TM system by BGI Europe (Copenhagen, Denmark). BWA V.0.78 2 and GATK HaplotypeCaller V.3.3 3 were used for read mapping along the hg19 reference genome (GRCh37/hg19) and variant calling, respectively. Variants were annotated using an in-house developed pipeline. WGS was performed by BGI (Hongkong, China) on a BGISeq500 using a 2x 100 bp paired end module, with a minimal median coverage per genome of 30-fold. SVs were called using Manta Structural Variant Caller V.1.1.0 (Illumina; paired end and split read evidence for SVs) and copy number variants (CNVs) using Control-FREEC (detection of copy number changes and allelic imbalances based on read depth). 4 Variants were validated and visualized using the IGV software (V.2.4). 5 Shared single nucleotide variants (SNVs) or SVs located in or spanning the refined RP17-locus were assessed for putative pathogenicity. Variants were prioritized based on a minor allele frequency (MAF) ≤0.0001 in gnomAD. 6 Index family UK1; WES was performed for one affected individual as previously described. 7 WGS was subsequently performed for four affected individuals from distant branches of the family by Edinburgh Genomics using TruSeq Nano with a minimal median coverage of 30-fold per genome. Variants were assessed and filtered using the Variant Annotation and Filter Tool (VarAFT). 8 Variants were prioritized based on a MAF